Method for the production of haploid and subsequent doubled haploid plants

ABSTRACT

It was found that plants with loss of functional Msi2 protein due to a nucleotide polymorphism resulting in the introduction of a premature stop codon in the Msi2 protein, are able to induce haploid offspring after a cross to or with a wild type plant comprising a functional Msi2 protein. The invention relates to generation of haploid and doubled haploid plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/763,710 filed Mar. 27, 2018 which is the national phase entry ofInternational Patent Application No. PCT/NL2016/050683, filed Oct. 3,2016, published on Apr. 6, 2017 as WO 2017/058023 A1, which claimspriority to Netherlands Patent Application No. 2015549, filed Oct. 2,2015. The contents of these applications are herein incorporated byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. The ASCII copy, created on Sep. 26, 2018, isnamed 085342-2401SequenceListing.txt and is 29 KB.

FIELD OF THE INVENTION

The disclosure relates to the field of agriculture. In particular, thedisclosure relates to the production of haploid and subsequent doubledhaploid plants.

BACKGROUND OF THE INVENTION

A high degree of heterozygosity in breeding material can make plantbreeding and selection for beneficial traits a very time consumingprocess. Extensive population screening, even with the latest molecularbreeding tools, is both laborious and costly.

The creation of haploid plants followed by chemical or spontaneousgenome doubling is an efficient way to solve the problem of highheterozygosity. Such doubled haploids bypass at least 7 generations ofselfing otherwise needed to reduce the heterozygosity to an acceptablelevel. Haploid plants can be produced in some crops by microsporeculture. However, this is costly and time-consuming. More importantly,in many crops microspore culture methods do not work. In some cropspecies, (doubled) haploid plants can be obtained by parthenogenesis ofthe egg cell or by elimination of one of the parental genomes. However,these methods are also restricted to a few selected crops and theproduction rates of doubled haploid plants are low.

WO2011/044132 discloses methods of producing haploid plants. One of themethods employed is inactivating or knocking out CenH3 protein. This wasdone by adding an N-terminal GFP to the CenH3 protein, thereby creatingGFP-CenH3. This is also called a “tailswap”. The tailswap was sufficientto induce uni-parental genome elimination upon a cross to a plantwithout such modified N-terminal part of the CenH3 protein. Theuni-parental genome elimination resulted in the production of a haploidplant. So far this process has only been demonstrated in the model plantArabidopsis thaliana and not in crop plants. Additionally, when anotherartificial construct, which consisted of a different trans-geneticallymodified N-terminal part of the CenH3 protein, was introduced in a plantwith a genetic background lacking the endogenous CenH3, it appeared thatthis did not resulted in uni-parental genome elimination and subsequentproduction of a haploid plant (WO 2014/110274). Therefore it remainselusive which modifications of the CenH3 protein are sufficient foruni-parental genome elimination.

Thus, there remains a need in the art for methods that allow efficientgeneration of haploid plants which can subsequently be doubled, toproduce doubled haploid plants. With doubled haploid production systems,homozygosity is achieved in one generation.

SUMMARY OF THE INVENTION

The present inventors have now found that Solanum lycopersicum plantswith loss of functional Msi2 protein due to a unique single nucleotidepolymorphism resulting in a K to STOP codon amino acid modification atposition 126 in the Solanum lycopersicum Msi2 protein, are able toinduce haploid offspring after a cross to or with a wild type plantcomprising a functional Msi2 protein. A single nucleotide polymorphismresulting in a K to M amino acid modification in the Msi2 protein wasfound to be non-disruptive by computational methods (SIFT, Kumar P,Henikoff S, Ng PC. (2009) Predicting the effects of codingnon-synonymous variants on protein function using the SIFT algorithm.Nat Protoc; 4(7):1073-81). In line with these models, the latter aminoacid modification did not induce haploid offspring after a cross to orwith a wild type plant lacking that particular K to M acid modificationin the Msi2 protein. Reciprocal control cross with a synonymous singlenucleotide polymorphism, not changing the amino acid sequence(Msi2D337D), did not yield any haploid offspring.

In a first aspect, the present invention pertains to an Msi2 protein ofplant origin comprising a loss-of-function mutation.

Said mutation may be present in a WD40 repeat and/or in a CAF1C domain.

Said Msi2 protein may be encoded by a plant Msi2 protein-encodingpolynucleotide having a loss-of-function mutation, which protein, whenpresent in a plant in the absence of its endogenous Msi2 protein, allowsgeneration of some haploid progeny, or progeny with aberrant ploidy,when said plant is crossed with a wild-type plant.

Said Msi2 protein may be derived from a polypeptide comprising the aminoacid sequence of SEQ ID NO:1 or 10, or a variant thereof having at least70%, more preferably at least 80%, even more preferably at least 90%,yet even more preferably at least 95%, most preferably at least 98% or99% sequence identity to the amino acid sequence of SEQ ID NO:1 or 10,said protein being encoded by a plant Msi2 protein-encodingpolynucleotide having a loss-of-function mutation, which protein, whenpresent in a plant in the absence of its endogenous Msi2 protein, allowsgeneration of some haploid progeny, or progeny with aberrant ploidy,when said plant is crossed with a wild-type plant.

Said Msi2 protein may be derived from a polypeptide comprising the aminoacid sequence of SEQ ID NO:2 or 3, or a variant thereof having at least70%, more preferably at least 80%, even more preferably at least 90%,yet even more preferably at least 95%, most preferably at least 98% or99% sequence identity to the amino acid sequence of SEQ ID NO:2 or 3,said protein being encoded by a plant Msi2 protein-encodingpolynucleotide having a loss-of-function mutation, which protein, whenpresent in a plant in the absence of its endogenous Msi2 protein, allowsgeneration of some haploid progeny, or progeny with aberrant ploidy,when said plant is crossed with a wild-type plant. For example, at least0.1, 0.5, 1 or 5% of the progeny produced is haploid, or has an aberrantploidy, or is doubled-haploid.

The loss-of-function mutation may introduce a premature stop codon thatcauses truncation of the protein. For example, the protein may betruncated after the amino acid residue at position 125 in SEQ ID NO:2, 3or 10, or after the amino acid residue at position 123 in SEQ ID NO:1.

In an embodiment, the Msi2 protein comprises the amino acid sequence ofSEQ ID NO:6 or consists of the amino acid sequence of SEQ ID NO:6.

In an embodiment, the Msi2 protein may be encoded by a nucleic acidmolecule comprising the nucleic acid sequence of SEQ ID NO:4 or 9.

The Msi2 protein may be encoded by a polynucleotide comprising aloss-of-function mutation that is derived from a polynucleotide encodingan endogenous Msi2 protein using targeted nucleotide exchange or byapplying an endonuclease.

In another aspect, the invention provides a nucleic acid moleculeencoding the Msi2 protein taught herein.

Also, the invention provides a nucleic acid molecule comprising thenucleic acid sequence of SEQ ID NO:5, 7, or 8, or a variant thereofhaving at least 70%, more preferably at least 80%, even more preferablyat least 90%, yet even more preferably at least 95%, most preferably atleast 98% or 99% sequence identity to the nucleic acid sequence of SEQID NO:5, 7 or 8, in which one or more nucleotides of the nucleic acidsequence of SEQ ID NO:5, 7 or 8 are modified such that the nucleic acidmolecule encodes a Msi2 protein comprising a loss-of-function mutation.

The invention also provides a nucleic acid molecule comprising thenucleic acid sequence of SEQ ID NO:5, 7, or 8, or a variant thereofhaving at least 70%, more preferably at least 80%, even more preferablyat least 90%, yet even more preferably at least 95%, most preferably atleast 98% or 99% sequence identity to the nucleic acid sequence of SEQID NO:5, 7, or 8, in which one or more nucleotides are modified suchthat a premature stop codon is introduced and the nucleic acid moleculeencodes a truncated Msi2 protein.

In an embodiment, one or more nucleotides at positions 376, 377 and/or378 of the nucleic acid sequence of SEQ ID NO:5 or 7, or one or morenucleotides at positions 685, 686 and/or 687 of the nucleic acidsequence of SEQ ID NO:8, are modified such that a stop codon isintroduced and the nucleic acid molecule encodes a polypeptidecomprising an amino acid sequence that is truncated after the amino acidresidue corresponding to position 125 in SEQ ID NO:2 or 3.

Also taught is a nucleic acid molecule comprising the nucleic acidsequence of SEQ ID NO:4 or 9.

The nucleic acid molecule taught herein may be an isolated nucleic acid,a genomic nucleic acid, or a cDNA.

The invention further provides a chimeric gene comprising the nucleicacid molecule taught herein, and a vector comprising the nucleic acidmolecule taught herein or the chimeric gene taught herein.

The invention further pertains to a host cell comprising a nucleic acidmolecule taught herein, a chimeric gene taught herein, or a vector astaught herein.

Said host cell may be a plant cell, including a protoplast, preferably atomato plant cell.

Also disclosed is a plant, seed, or plant cell comprising the nucleicacid molecule as taught herein, a chimeric gene as taught herein, or avector as taught herein.

In an embodiment, the endogenous Msi2 protein is not expressed in saidplant, seed, or plant cell.

The invention also relates to a plant, seed, or plant cell wherein theendogenous Msi2 protein is not expressed, for example, wherein theendogenous Msi2 gene is knocked out. In an embodiment, said plant, seed,or plant cell is not an Arabidopsis thaliana plant, seed, or plant cell.

Said plant, seed, or plant cell may be a Solanum plant, seed, or plantcell, preferably a Solanum lycopersicum plant, seed, or plant cell.

The invention is further concerned with a method for making a plant,seed, or plant cell as taught herein, said method comprising the stepsof:

-   a) modifying an endogenous Msi2 gene within a plant cell to obtain a    mutated Msi2 gene encoding an Msi2 protein as taught herein, or    modifying an endogenous Msi2 gene within a plant cell in order to    knock out expression of endogenous Msi2 protein within a plant cell;-   b) selecting a plant cell comprising the mutated Msi2 gene, or a    plant cell in which expression of said endogenous Msi2 protein is    knocked out; and-   c) optionally, regenerating a plant from said plant cell.

The invention is also directed to a method for making a plant, seed, orplant cell as taught herein, said method comprising the steps of:

-   a) transforming a plant cell with a nucleic acid molecule as taught    herein, a chimeric gene as taught herein, or a vector as taught    herein, or transforming a plant cell with a nucleic acid molecule in    order to knock out expression of an endogenous Msi2 protein;-   b) optionally, additionally modifying an endogenous Msi2 gene within    a plant cell in order to knock out expression of endogenous Msi2    protein within said plant cell;-   c) selecting a plant cell comprising the nucleic acid molecule as    taught herein, a chimeric gene as taught herein, or a vector as    taught herein, and/or a plant cell in which expression of endogenous    Msi2 protein is knocked out; and-   d) optionally, regenerating a plant from said plant cell.

In another aspect, the invention provides for a method of generating ahaploid plant, a plant with aberrant ploidy, or a doubled haploid plant,said method comprising the steps of:

-   a) crossing a plant expressing an endogenous Msi2 protein to the    plant as taught herein, wherein the plant as taught herein lacks    expression of endogenous Msi2 protein at least in its reproductive    parts and/or during embryonic development;-   b) harvesting seed;-   c) growing at least one seedling, plantlet or plant from said seed;    and-   d) selecting a haploid seedling, plantlet or plant, a seedling,    plantlet or plant with aberrant ploidy, or a doubled haploid    seedling, plantlet or plant.

The invention also relates to a method of generating a doubled haploidplant, said method comprising the steps of:

-   a) crossing a plant expressing an endogenous Msi2 protein to the    plant as taught herein, wherein the plant as taught herein lacks    expression of endogenous Msi2 protein at least in its reproductive    parts and/or during embryonic development;-   b) selecting a haploid plant; and-   c) converting said haploid plant into a doubled haploid plant.

The conversion in step c) may be performed by treatment with colchicine.

The plant expressing an endogenous Msi2 protein may be an F1 plant.

The plant expressing an endogenous Msi2 protein may be a pollen parentof the cross, or may be an ovule parent of the cross.

The cross may be performed at a temperature in the range of about 24 toabout 30° C., preferably in the range of about 26 to about 28° C.

The invention further relates to use of the nucleic acid molecule astaught herein for producing a haploid inducer line.

Also, the invention is directed to a Solanum lycopersicum plant, seed,or plant cell comprising the nucleic acid molecule as taught herein, thechimeric gene as taught herein, or the vector as taught herein.

Further, a Solanum lycopersicum plant, seed, or plant cell comprising anucleic acid molecule encoding a polypeptide comprising the amino acidsequence of SEQ ID NO:6, is taught herein.

Also, a Solanum lycopersicum plant, seed, or plant cell comprising thenucleic acid molecule of SEQ ID NO:4 or 9 is taught herein.

In yet another aspect, a Solanum lycopersicum plant, seed or plant cellcomprising a nucleic acid molecule that encodes a Msi2 protein as taughtherein, is provided.

Also, a Solanum lycopersicum plant, seed, or plant cell which lacksexpression of functional Msi2 protein; optionally, wherein an Msi2protein comprising a loss-of-function mutation is expressed, is taughtherein.

The Solanum lycopersicum plant as taught herein may be used forproducing a haploid Solanum lycopersicum plant, and/or for producing adoubled haploid Solanum lycopersicum plant.

The Solanum lycopersicum plant taught herein may lack expression ofendogenous Msi2 protein at least in its reproductive parts and/or duringembryonic development.

In a final aspect, the invention is directed to a method of generating ahaploid or doubled haploid plant, said method comprising the step ofidentifying a plant expressing an endogenous Msi2 protein and a plant astaught herein, wherein the plant as taught herein lacks expression ofendogenous Msi2 protein at least in its reproductive parts and/or duringembryonic development.

The crossing methods as taught herein do not comprise sexually crossingthe whole genomes of said plants.

Definitions

The term “Msi2” refers to Musashi RNA-binding protein 2. It is a WD-40repeat-containing protein. WD-40 repeats (also known as WD orbeta-transducin repeats) are short ˜40 amino acid motifs, oftenterminating in a Trp-Asp (W-D) dipeptide. WD40 repeats usually assume a7-8 bladed beta-propeller fold, but proteins have been found with 4 to16 repeated units, which also form a circularized beta-propellerstructure. WD-repeat proteins are a large family found in all eukaryotesand are implicated in a variety of functions ranging from signaltransduction and transcription regulation to cell cycle control andapoptosis. Repeated WD40 motifs act as a site for protein-proteininteraction, and proteins containing WD40 repeats are known to serve asplatforms for the assembly of protein complexes or mediators oftransient interplay among other proteins. The specificity of theproteins is determined by the sequences outside the repeats themselves.Msi2 is thought to be an RNA binding protein that regulates theexpression of target mRNAs at the translation level. In Arabidopsisspp., several WD40-containing proteins act as key regulators ofplant-specific developmental events. An Arabidopsis thaliana Msi2(At2g16780) T-DNA insertion mutant and Msi2-overexpressing transgenicplant were reported to have the same phenotype as wild-type. Theexpression of the Msi2 gene in wild-type could not be induced bydrought, salt, high temperature, low temperatures, mannitol, ABA, GA,salicylic acid and jasmonic acid. A localization study using a fusionprotein consisting of the full length of Msi2 cDNA and GFP under thecontrol of 35S promoter revealed that fluorescence was detected in boththe cytosol and nucleus. The exact role of Msi2 in the plant is yetunknown.

A “mutation” is a permanent change of the nucleotide sequence of thegenome of an organism, virus, or extrachromosomal DNA or other geneticelements. Mutations result from damage to DNA which is not repaired orto RNA genomes (typically caused by radiation or chemical mutagens),errors in the process of replication, or from the insertion or deletionof segments of DNA by mobile genetic elements. Mutations may or may notproduce discernible changes in the observable characteristics(phenotype) of an organism. A mutation can result in several differenttypes of change in sequences. Mutations in genes can either have noeffect, alter the product of a gene, or prevent the gene fromfunctioning properly or completely. Mutations can also occur innon-genic regions.

A “loss-of-function mutation” in a protein in the context of the presentinvention refers to a mutation in a protein that causes loss of itsfunction. A “loss-of-function mutation” in a polynucleotide refers to amutation in a polynucleotide encoding a protein that causesloss-of-function of said protein. A protein may still be produced fromthe polynucleotide comprising the loss-of-function mutation, but theprotein can no longer perform its function or cannot perform itsfunction effectively.

A “loss-of-expression mutation” results in loss of expression of themutated gene or the product encoded by said mutated gene.

A “loss-of-function Msi2 protein” is an Msi2 protein that comprises aloss-of-function mutation. Said loss-of-function Msi2 protein, whenpresent in a plant in the absence of its endogenous Msi2 protein, allowsgeneration of some haploid progeny, or progeny with aberrant ploidy,when said plant is crossed with a wild-type plant, preferably awild-type plant of the same species. A loss-of-function Msi2 protein mayalso become non-functional or less functional by using inhibitors of theprotein, such as an antibody specifically binding the Msi2 protein, orother Msi2 inhibitors, e.g. proteins that block, prevent or reduce theactivity of an endogeous Msi2 protein, or chemical inhibitors such asions, or metals, or scavenging of co-factors. For example, an antibodyspecifically binding Msi2 protein may be expressed simultaneously withsaid Msi2 protein, thereby reducing its specific activity. The Msi2protein function may be impaired, or the Msi2 protein may be lessfunctional than the endogenous Msi2 protein.

A “loss-of-function Msi2 protein-encoding polynucleotide” refers to anon-endogenous, mutated Msi2 protein-encoding polynucleotide thatencodes an Msi2 protein comprising a loss-of-function mutation, which,when present in a plant in the absence of its endogenous Msi2protein-encoding polynucleotide, allows generation of some haploidprogeny, or progeny with aberrant ploidy, when said plant is crossedwith a wild-type plant, preferably a wild-type plant of the samespecies. A loss-of-function mutation includes a frame-shift mutation.

The term “endogenous” as used in the context of the present invention incombination with protein, gene, or polynucleotide means that saidprotein, gene or polynucleotide originates from the plant in which it isstill contained. Often an endogenous gene will be present in its normalgenetic context in the plant.

The term “gene” as used herein refers to a DNA sequence comprising aregion (transcribed region), which is transcribed into an RNA molecule(e.g. an mRNA) in a cell, operably linked to suitable regulatory regions(e.g. a promoter). A gene may thus comprise several operably linkedsequences, such as a promoter, a 5′ leader sequence comprising e.g.sequences involved in translation initiation, a (protein) coding region(cDNA or genomic DNA) and a 3′ non-translated sequence comprising e.g.transcription termination sites.

The term “haploid inducer line” used in the context of the presentdisclosure refers to a plant line which differs in at least one singlenucleotide polymorphism from the non-inducer line. When an haploidinducer line is crossed, either used as female or as pollen donor, itresults in uni-parental genome elimination of the haploid inducer line'sgenome.

The term “uni-parental genome elimination” as used herein refers to theeffect of losing all the genetic information, meaning all chromosomes,of one parent after a cross irrespective of the direction of the cross.This occurs in such way that the offspring of such cross will onlycontain chromosomes of the non-eliminated parental genome. The genomewhich is eliminated always has the origin in the haploid inducer parent.

A “doubled haploid” is a genotype formed when haploid cells undergochromosome doubling. It may be produced by induced or spontaneouschromosome doubling from haploid cells. For diploid plants, the haploidcells are monoploid, and the term “doubled monoploid” may also be usedfor the doubled haploids.

A “frame-shift mutation” (also called a framing error or a reading frameshift) is a genetic mutation caused by indels (insertions or deletions)of a number of nucleotides that is not evenly divisible by three in anucleotide sequence. Due to the triplet nature of gene expression bycodons, the insertion or deletion can change the reading frame (thegrouping of the codons), resulting a completely different translation ofthe template. The earlier in the sequence the deletion or insertionoccurs, the more altered the protein product is. A frame shift mutationwill in general cause the reading of the codons after the mutation tocode for different amino acids, but there may be exceptions resultingfrom the redundancy in the genetic code. Furthermore, the stop codon inthe original sequence will not be read, and an alternative stop codonmay result at an earlier or later stop site. The protein product may beabnormally short or abnormally long.

The terms “polynucleotide”, “nucleic acid molecule”, and “nucleic acid”are used interchangeably herein.

A “chimeric gene” (or recombinant gene) refers to any gene, which is notnormally found in nature in a species, in particular a gene in which oneor more parts of the nucleic acid sequence are present that are notassociated with each other in nature. For example the promoter is notassociated in nature with part or all of the transcribed region or withanother regulatory region. The term “chimeric gene” is understood toinclude expression constructs in which a promoter or transcriptionregulatory sequence is operably linked to one or more coding sequencesor to an antisense (reverse complement of the sense strand) or invertedrepeat sequence (sense and antisense, whereby the RNA transcript formsdouble stranded RNA upon transcription).

“Sequence identity” and “sequence similarity” can be determined byalignment of two peptide or two nucleotide sequences using global orlocal alignment algorithms. Sequences may then be referred to as“substantially identical” or “essentially similar” when they (whenoptimally aligned by for example the programs GAP or BESTFIT usingdefault parameters) share at least a certain minimal percentage ofsequence identity (as defined below). GAP uses the Needleman and Wunschglobal alignment algorithm to align two sequences over their entirelength, maximizing the number of matches and minimises the number ofgaps. Generally, the GAP default parameters are used, with a gapcreation penalty=50 (nucleotides)/8 (proteins) and gap extensionpenalty=3 (nucleotides)/2 (proteins). For nucleotides the defaultscoring matrix used is nwsgapdna and for proteins the default scoringmatrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919).Sequence alignments and scores for percentage sequence identity may bedetermined using computer programs, such as the GCG Wisconsin Package,Version 10.3, available from Accelrys Inc., 9685 Scranton Road, SanDiego, Calif. 92121-3752 USA, or EmbossWin version 2.10.0 (using theprogram “needle”). Alternatively percent similarity or identity may bedetermined by searching against databases, using algorithms such asFASTA, BLAST, etc.

A “host cell” or a “recombinant host cell” or “transformed cell” areterms referring to a new individual cell (or organism) arising as aresult of introduction of at least one nucleic acid molecule, especiallycomprising a chimeric gene encoding a desired protein. The host cell ispreferably a plant cell or a bacterial cell. The host cell may containthe nucleic acid molecule or chimeric gene as an extra-chromosomally(episomal) replicating molecule, or more preferably, comprises thenucleic acid molecule or chimeric gene integrated in the nuclear orplastid genome of the host cell.

As used herein, the term “plant” includes plant cells, plant tissues ororgans, plant protoplasts, plant cell tissue cultures from which plantscan be regenerated, plant calli, plant cell clumps, and plant cells thatare intact in plants, or parts of plants, such as embryos, pollen,ovules, fruit (e.g. harvested tomatoes), flowers, leaves, seeds, roots,root tips and the like.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. It encompasses the verbs “to essentially consist of”and “to consist of”.

In addition, reference to an element by the indefinite article “a” or“an” does not exclude the possibility that more than one of the elementis present, unless the context clearly requires that there be one andonly one of the elements. The indefinite article “a” or “an” thususually means “at least one”. It is further understood that, whenreferring to “sequences” herein, generally the actual physical moleculeswith a certain sequence of subunits (e.g. amino acids) are referred to.

DETAILED DESCRIPTION OF THE INVENTION Mode of Action of the Invention

It is believed that plants in which expression of functional Msi2protein is impaired and/or functionality of the Msi2 protein is impairedresulting in the absence or reduced presence of functional Msi2 protein,gives such plant the haploid inducer phenotype. When said plant iscrossed with a sexually compatible wild-type plant, preferably awild-type plant of the same species, generation of some haploid progeny,or progeny with aberrant ploidy, is induced at relatively highfrequency. The percentage of haploid progeny or progeny with aberrantploidy that is generated using such plant can, for instance, be at least0.1, 0.5, 1, 5, 10, 20%, or more.

Impaired expression of a functional Msi2 protein may mean that theexpression of the Msi2 gene is impaired, and/or that expression of theMsi2 gene is normal but translation of the transcribed mRNA is inhibitedor prevented (for example by RNA interference).

Impaired expression of functional Msi2 protein at the transcriptionallevel can be the result of the introduction of one or more mutations intranscription regulation sequences, including promoters, enhancers, orinitiation, termination or intron splicing sequences. These sequencesare generally located 5′ of, or 3′ of, or within the coding sequence ofthe gene or genes coding for Msi2 protein. Alternatively oradditionally, impaired expression of functional Msi2 protein can also beachieved by deletion, substitution, rearrangement or insertion ofnucleotides in the coding region of the endogenous Msi2 gene or genes.For example, in the coding region, nucleotides may be substituted,inserted or deleted leading to the introduction of one, two or morepremature stop codons. Also, insertion, deletion, rearrangement orsubstitution can lead to modifications in the amino acid sequenceencoded, and thereby provide for impaired expression of functional Msi2protein. Large parts of the Msi2 gene(s) may be removed, for example, atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100% of the(coding region) of the Msi2 gene(s) may be removed from the DNA presentin the plant, thereby impairing expression of functional Msi2 protein.

Alternatively, one, two, three or more nucleotides may be introducedinto the Msi2 gene(s), leading to, for example, a frame-shift, or to theintroduction of a additional amino acids into the Msi2 protein, or tothe introduction of nucleic acid sequence not encoding amino acids, orthe introduction of large inserts (e.g., T-DNA insertion), therebyimpairing the provision/expression of functional Msi2 protein.

Impairment at the translational level can be achieved by theintroduction of a premature stop codon or by influencing other RNA toprotein processing mechanisms (such as splicing) or post-translationalmodification influencing, for example, protein folding or cellulartrafficking.

Impairment or loss-of-function at the protein level can be provided bytruncation, or by modification of amino acid residues important foractivity, substrate binding, co-factor binding, folding, protein-proteininteractions, and the like.

Impairment of expression of functional Msi2 protein may also beaccomplished by gene silencing, for example, using CRISP, RNAi, VIGS, orthe like.

Additionally, impairment of expression of functional Msi2 protein may beachieved by using Msi2 protein inhibitors such as an antibodyspecifically binding the Msi2 protein, or other Msi2 inhibitors, e.g.proteins that block, prevent or reduce the activity of endogenous Msi2protein, or chemical inhibitors such as ions, or metals, or scavengingof co-factors. For example, an antibody specifically binding Msi2protein may be expressed simultaneously with said Msi2 protein, therebyreducing its specific activity.

Msi2 Protein Comprising a Loss-of-Function Mutation

The present invention provides an Msi2 protein, preferably of plantorigin, comprising one or more loss-of-function mutations. When a plantthat expresses such Msi2 protein comprising one or more loss-of-functionmutations and lacks expression of, or has suppressed expression of,endogenous Msi2 protein, is crossed to a wild type plant expressingendogenous Msi2 protein, haploid plants are formed at relatively highfrequency. Haploid plants are formed at a more than normal frequency,such as at least 0.1, 0.5, 1, 5, 10, 20%, or more. Msi2 proteincomprising one or more loss-of-function mutations can be created by avariety of means known to the skilled person. These include, withoutlimitation, random mutagenesis, single or multiple amino acid targetedmutagenesis, generation of complete or partial protein domain deletions,fusion with heterologous amino acid sequences, and the like. Typically,the polynucleotide encoding endogenous Msi2 protein will be knocked outor inactivated to create a plant that lacks expression of endogenousMsi2 protein.

Msi2 protein comprising one or more loss-of-function mutations can, forexample, be tested by recombinant expression of the Msi2 proteincomprising one or more loss-of-function mutations in a plant lackingexpression of endogenous Msi2 protein, crossing the transgenic plant toa plant expressing endogenous Msi2 protein, and then screening for theproduction of haploid progeny.

Any number of mutations can be introduced into an endogenous Msi2protein to generate an Msi2 protein comprising one or moreloss-of-function mutations. For example, the Msi2 protein comprising oneor more loss-of-function mutations may be identical to the endogenousMsi2 protein but for 1, 2, 3, 4, 5, 6, 7, 8, or more amino acids.

The Msi2 protein is preferably a plant Msi2 protein. The plant may beany plant, but preferably belongs to the Solanaceae family, morepreferably to the genus Solanum, even more preferably to the speciesSolanum lycopersicum.

In an embodiment, the one or more loss-of-function mutations are made inthe endogenous Msi2 protein as represented by an amino acid sequence asshown in any of SEQ ID NOs: 1, 10, 2, or 3. Alternatively oradditionally, the one or more loss-of-function mutations may be made inthe endogenous Msi2 protein as represented by an amino acid sequence asshown in any of SEQ ID NOs: 1, 10, 2, or 3, or a variant thereof havingat least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more, such as 100%, amino acid sequence identity to theamino acid sequence of any of SEQ ID NOs: 1, 10, 2, or 3, preferablyover the entire length. Amino acid sequence identity is determined bypairwise alignment using the Needleman and Wunsch algorithm and GAPdefault parameters as defined above.

The one or more loss-of-function mutations within the Msi2 proteincomprising one or more loss-of-function mutations may be locatedthroughout the protein. In an embodiment, a loss-of-function mutation islocated in a WD40 repeat or in a CAF1C domain of the endogenous Msi2protein as represented by an amino acid sequence as shown in any of SEQID NO: 1, 10, 2, or 3, or a variant thereof as taught herein.

The Msi2 protein taught herein may be encoded by a plant Msi2protein-encoding polynucleotide having a loss-of-function mutation. Theprotein, when present in a plant in the absence of its endogenous Msi2protein, allows generation of some haploid progeny, or progeny withaberrant ploidy, when said plant is crossed with a wild-type plant,without affecting viability of said plant.

The Msi2 protein taught herein may be derived from the a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or 10, or a variantthereof having at least 70%, more preferably at least 80%, even morepreferably at least 90%, yet even more preferably at least 95%, mostpreferably at least 98% or 99% sequence identity to the amino acidsequence of SEQ ID NO:1 or 10, said protein being encoded by a plantMsi2 protein-encoding polynucleotide having a loss-of-function mutation,which protein, when present in a plant in the absence of its endogenousMsi2 protein, allows generation of some haploid progeny, or progeny withaberrant ploidy, when said plant is crossed with a wild-type plant.

The Msi2 protein taught herein may be derived from a polypeptidecomprising the amino acid sequence of SEQ ID NO:2 or 3, or a variantthereof having at least 70%, more preferably at least 80%, even morepreferably at least 90%, yet even more preferably at least 95%, mostpreferably at least 98% or 99% sequence identity to the amino acidsequence of SEQ ID NO:2, said polypeptide being encoded by a plant Msi2protein-encoding polynucleotide having a loss-of-function mutation,which polypeptide, when present in a plant in the absence of itsendogenous Msi2 protein, allows generation of some haploid progeny, orprogeny with aberrant ploidy, when said plant is crossed with awild-type plant.

The Msi2 protein taught herein may be derived from the polypeptidecomprising the amino acid sequence of SEQ ID NO:3 or a variant thereofhaving at least 70%, more preferably at least 80%, even more preferablyat least 90%, yet even more preferably at least 95%, most preferably atleast 98% or 99% sequence identity to the amino acid sequence of SEQ IDNO:3, said polypeptide being encoded by a plant Msi2 protein-encodingpolynucleotide having loss-of-function mutation, which, when present ina plant in the absence of its endogenous Msi2 protein, allows generationof some haploid progeny, or progeny with aberrant ploidy, when saidplant is crossed with a wild-type plant.

Suitably, at least 0.1, 0.5, 1 or 5% of the progeny produced when aplant as taught herein is crossed with a wild-type plant, is haploid, orhas an aberrant ploidy, or is doubled-haploid.

In an embodiment, the one or more loss-of-function mutations introduce apremature stop codon that causes truncation of the protein.

In one embodiment, the protein is truncated after the amino acid residueat position 125 in SEQ ID NO:2 or 3.

In an embodiment, the Msi2 protein taught herein is encoded by a nucleicacid molecule comprising the nucleic acid sequence of SEQ ID NO:4 or 9.

Polynucleotides Encoding an Msi2 Protein Comprising One or MoreLoss-of-Function Mutations, Chimeric Genes, Vectors, Host Cells

Polynucleotides having nucleic acid sequences, such as cDNA, genomic DNAand RNA molecules, encoding any of the above proteins are also provided.Due to the degeneracy of the genetic code a variety of nucleic acidsequences may encode the same amino acid sequence. Any polynucleotidesencoding Msi2 proteins or variants thereof are herein referred to as“Msi2 protein-encoding polynucleotides”. The polynucleotides providedinclude naturally occurring, artificial or synthetic nucleic acidsequences. It is understood that when sequences are depicted as DNAsequences while RNA is referred to, the actual base sequence of the RNAmolecule is identical with the difference that thymine (T) is replace byuracil (U).

The present invention further relates to a polynucleotide encoding aMsi2 protein comprising one or more loss-of-function mutations as taughtherein. Said polynucleotide may be a synthetic, recombinant and/orisolated polynucleotide. In an embodiment, said polynucleotide isderived from an endogenous Msi2 protein-encoding polynucleotide, e.g.,an Msi2 gene, that comprises the nucleic acid sequence of SEQ ID NO:5,7, or 8, or a variant thereof having at least 70%, preferably at least75%, such as 80%, 85%, 90%, 95%, more preferably at least 97%, 98%, or99% sequence identity to the nucleic acid sequence of SEQ ID NO: 5, 7,or 8, preferably over the full length, and which shares the endogenousMsi2 activity of the polypeptide comprising the amino acid sequence ofSEQ ID NO:5, 7, or 8. In contrast, the Msi2 protein-encodingpolynucleotide comprising one or more loss-of-function mutations taughtherein comprises one or more mutations that reduce(s) or eliminate(s)endogenous Msi2 activity to less than 90, 80, 70, 60, 50, 40, 30, 20,10% of Msi2 activity of endogenous Msi2 protein. Preferably, whenpresent in a plant in the absence of the endogenous Msi2protein-encoding polynucleotide, the Msi2 protein taught herein allowsgeneration of some haploid progeny, or progeny with aberrant ploidy,when said plant is crossed with a wild-type plant.

The nucleic acid molecule as taught herein may be used for producing ahaploid inducer line.

In one embodiment of the invention, nucleic acid sequences encoding Msi2proteins (including Msi2 proteins comprising one or moreloss-of-function mutations, or variants or fragments thereof), asdescribed above, are used to make chimeric genes, and/or vectors fortransfer of the Msi2 protein-encoding polynucleotides into a host celland production of the Msi2 protein(s) in host cells, such as cells,tissues, organs or organisms derived from transformed cell(s). Vectorsfor the production of Msi2 protein (or protein fragments or variantsthereof) as taught herein in plant cells are herein referred to as“expression vectors”.

Suitable host cells for expression of Msi2 proteins include prokaryotes,yeast, or higher eukaryotic cells. Appropriate cloning and expressionvectors for use with bacterial, fungal, yeast, and mammalian cellularhosts are described, for example, in Pouwels et al., Cloning vectors: ALaboratory Manual, Elsevier, N.Y., (1985). Cell-free translation systemscould also be employed to produce the proteins of the present inventionusing RNAs derived from nucleic acid sequences disclosed herein.

Suitable prokaryotic host cells include gram-negative and gram-positiveorganisms, for example, Escherichia coli or Bacilli. Another suitableprokaryotic host cell is Agrobacterium, in particular Agrobacteriumtumefaciens.

Msi2 proteins as taught herein can also be expressed in yeast hostcells, for example from the Saccharomyces genus (e.g., Saccharomycescerevisiae). Other yeast genera, such as Pichia or Kluyveromyces, canalso be employed.

Alternatively, Msi2 proteins as taught herein may be expressed in highereukaryotic host cells, including plant cells, fungal cells, insectcells, and mammalian, optionally non-human, cells.

One embodiment of the invention is a non-human organism modified tocomprise a polynucleotide as taught herein. The non-human organismand/or host cell may be modified by any methods known in the art forgene transfer including, for example, the use of delivery devices suchas lipids and viral vectors, naked DNA, electroporation, chemicalmethods and particle-mediated gene transfer. In an advantageousembodiment, the non-human organism is a plant.

Any plant cell may be a suitable host cell. The term “plant cell” asused herein includes protoplasts. Suitable plant cells include thosefrom monocotyledonous plants or dicotyledonous plants. For example, theplant may belong to the genus Solanum (including Lycopersicon),Nicotiana, Capsicum, Petunia and other genera. The following hostspecies may suitably be used: Tobacco (Nicotiana species, e.g. N.benthamiana, N. plumbaginifolia, N. tabacum, etc.), vegetable species,such as tomato (L. esculentum, syn. Solanum lycopersicum) such as e.g.cherry tomato, var. cerasiforme or currant tomato, var.pimpinellifolium) or tree tomato (S. betaceum, syn. Cyphomandrabetaceae), potato (Solanum tuberosum), eggplant (Solanum melongena),pepino (Solanum muricatum), cocona (Solanum sessiliflorum) andnaranjilla (Solanum quitoense), peppers (Capsicum annuum, Capsicumfrutescens, Capsicum baccatum), ornamental species (e.g. Petuniahybrida, Petunia axillaries, P. integrifolia), coffee (Coffea).

Alternatively, the plant may belong to any other family, such as to theCucurbitaceae or Gramineae. Suitable host plants include for examplemaize/corn (Zea species), wheat (Triticum species), barley (e.g. Hordeumvulgare), oat (e.g. Avena sativa), sorghum (Sorghum bicolor), rye(Secale cereale), soybean (Glycine spp, e.g. G. max), cotton (Gossypiumspecies, e.g. G. hirsutum, G. barbadense), Brassica spp. (e.g. B. napus,B. juncea, B. oleracea, B. rapa, etc), sunflower (Helianthus annus),safflower, yam, cassava, alfalfa (Medicago sativa), rice (Oryza species,e.g. O. sativa indica cultivar-group or japonica cultivar-group), foragegrasses, pearl millet (Pennisetum spp. e.g. P. glaucum), tree species(Pinus, poplar, fir, plantain, etc), tea, coffea, oil palm, coconut,vegetable species, such as pea, zucchini, beans (e.g. Phaseolusspecies), cucumber, artichoke, asparagus, broccoli, garlic, leek,lettuce, onion, radish, turnip, Brussels sprouts, carrot, cauliflower,chicory, celery, spinach, endive, fennel, beet, fleshy fruit bearingplants (grapes, peaches, plums, strawberry, mango, apple, plum, cherry,apricot, banana, blackberry, blueberry, citrus, kiwi, figs, lemon, lime,nectarines, raspberry, watermelon, orange, grapefruit, etc.), ornamentalspecies (e.g. Rose, Petunia, Chrysanthemum, Lily, Gerbera species),herbs (mint, parsley, basil, thyme, etc.), woody trees (e.g. species ofPopulus, Salix, Quercus, Eucalyptus), fibre species e.g. flax (Linumusitatissimum) and hemp (Cannabis sativa), or model organisms, such asArabidopsis thaliana.

Preferred host cells are derived from “crop plants” or “cultivatedplants”, i.e. plant species which is cultivated and bred by humans. Acrop plant may be cultivated for food or feed purposes (e.g. fieldcrops), or for ornamental purposes (e.g. production of flowers forcutting, grasses for lawns, etc.). A crop plant as defined herein alsoincludes plants from which non-food products are harvested, such as oilfor fuel, plastic polymers, pharmaceutical products, cork, fibres (suchas cotton) and the like.

The construction of chimeric genes and vectors for, preferably stable,introduction of Msi2 protein-encoding nucleic acid sequences as taughtherein into the genome of host cells is generally known in the art. Togenerate a chimeric gene the nucleic acid sequence encoding a Msi2protein as taught herein is operably linked to a promoter sequence,suitable for expression in the host cells, using standard molecularbiology techniques. The promoter sequence may already be present in avector so that the Msi2 protein-encoding nucleic acid sequence is simplyinserted into the vector downstream of the promoter sequence. The vectormay then be used to transform the host cells and the chimeric gene maybe inserted in the nuclear genome or into the plastid, mitochondrial orchloroplast genome and expressed using a suitable promoter (e. g., McBride et al., 1995 Bio/Technology 13, 362; U.S. Pat. No. 5,693,507). Inan embodiment the chimeric gene as taught herein comprises a suitablepromoter for expression in plant cells or microbial cells (e.g.bacteria), operably linked to a nucleic acid sequence encoding a Msi2protein as taught herein, optionally followed by a 3′nontranslatednucleic acid sequence. The bacteria may subsequently be used for planttransformation (Agrobacterium-mediated plant transformation).

The present invention also relates to plants, particularly crop plants,more particularly plants belonging to the family Solanaceae, moreparticularly to the genus Solanum, yet more particularly to the speciesSolanum lycopersicum, which lack expression of functional Msi2 protein,either due to: 1) prevention or reduction of expression of the Msi2gene, e.g., by knocking out the Msi2 gene; 2) prevention or reduction oftranslation of mRNA transcribed from the Msi2 gene; or 3) the expressionof a non-functional Msi2 protein.

Plants Expressing Msi2 Polypeptides Comprising One or MoreLoss-of-Function Mutations

The present invention provides plants, seeds, or plant cells expressinga Msi2 polypeptide comprising one or more loss-of-function mutations astaught herein. The present invention also provides plants, seed, orplant cells comprising a polynucleotide as taught herein, a chimericgene as taught herein, or a vector as taught herein. The plant, seed, orplant cell preferably belongs to the family Solanaceae, more preferablyto the genus Solanum, yet more preferably to the species Solanumlycopersicum.

The plants, seeds, or plant cells preferably do not express, or expressat reduced levels (e.g., less than 90, 80, 70, 60, 50, 40, 30, 20, 10%of wild type levels), an endogenous Msi2 protein. For example, one cangenerate a mutation in an endogenous Msi2 protein that reduces oreliminates endogenous Msi2 protein activity or expression, or one cangenerate a knockout for endogenous Msi2 protein. In this case, one maygenerate a plant heterozygous for the gene knockout or mutation andintroduce an expression vector for expression of a Msi2 proteincomprising one or more loss-of-function mutations as taught herein inthe plant. Progeny from the heterozygote can then be selected that arehomozygous for the mutation or knockout but that comprise the Msi2protein comprising one or more loss-of-function mutations.

Accordingly, in plants, seeds, or plant cells taught herein preferablyone or both endogenous Msi2 alleles are knocked out or mutated such thatsaid plants or plant cells significantly or essentially completely lackendogenous Msi2 activity. It was found that such plants are viable. Inplants having more than a diploid set of chromosomes, all endogenousMsi2 alleles may be inactivated, mutated or knocked out. Alternatively,the expression of endogenous Msi2 protein may be silenced by any wayknown in the art, e.g. by introducing a siRNA or microRNA that reducesor eliminates expression of endogenous Msi2 protein.

In an embodiment, the invention pertains to a Solanum lycopersicumplant, seed, or plant cell comprising the nucleic acid molecule astaught herein.

In an embodiment, the invention pertains to a Solanum lycopersicumplant, seed, or plant cell comprising a nucleic acid molecule thatencodes a Msi2 protein as taught herein.

In an embodiment, said Solanum lycopersicum plant, seed, or plant cellcomprises a nucleic acid molecule encoding a polypeptide comprising theamino acid sequence of SEQ ID NO:6.

In an embodiment, said Solanum lycopersicum plant, seed, or plant cellcomprises the nucleic acid molecule of SEQ ID NO:4 or 9.

Said Solanum lycopersicum plant, seed, or plant cell may be used forproducing a haploid Solanum lycopersicum plant, and/or for producing adoubled haploid Solanum lycopersicum plant.

In an embodiment, said Solanum lycopersicum plant, seed, or plant celllacks expression of endogenous Msi2 protein at least during embryonicdevelopment.

Methods for the Generation of Plants

It is an embodiment of the invention to modify an endogenous Msi2 geneusing targeted mutagenesis methods (also referred to as targetednucleotide exchange (TNE) or oligo-directed mutagenesis (ODM)). Targetedmutagenesis methods include, without limitation, those employing zincfinger nucleases, Cas9-like, Cas9/crRNA/tracrRNA or Cas9/gRNA CRISPRsystems, or targeted mutagenesis methods employing mutagenicoligonucleotides, possibly containing chemically modified nucleotidesfor enhancing mutagenesis with sequence complementarity to the Msi2gene, into plant protoplasts (e.g., KeyBase® or TALENs).

Alternatively, mutagenesis systems such as TILLING (Targeting InducedLocal Lesions IN Genomics; McCallum et al., 2000, Nat Biotech 18:455,and McCallum et al. 2000, Plant Physiol. 123, 439-442, both incorporatedherein by reference) may be used to generate plant lines which comprisea Msi2 gene encoding a Msi2 protein comprising one or moreloss-of-function mutations. TILLING uses traditional chemicalmutagenesis (e.g. EMS mutagenesis) followed by high-throughput screeningfor mutations. Thus, plants, seeds and tissues comprising a Msi2 genehaving the desired mutation may be obtained.

The method may comprise the steps of mutagenizing plant seeds (e.g. EMSmutagenesis), pooling of plant individuals or DNA, PCR amplification ofa region of interest, heteroduplex formation and high-throughputdetection, identification of the mutant plant, sequencing of the mutantPCR product. It is understood that other mutagenesis and selectionmethods may equally be used to generate such modified plants. Seeds may,for example, be radiated or chemically treated and the plants may bescreened for a modified phenotype.

Modified plants may be distinguished from non-modified plants, i.e.,wild type plants, by molecular methods, such as the mutation(s) presentin the DNA, and by the modified phenotypic characteristics. The modifiedplants may be homozygous or heterozygous for the mutation.

Thus, a method for making a plant as taught herein is provided, saidmethod comprising the steps of:

-   a) modifying a nucleic acid molecule encoding an endogenous Msi2    protein within a plant cell to obtain a mutated nucleic acid    molecule encoding an Msi2 protein as taught herein, or modifying a    nucleic acid molecule encoding an endogenous Msi2 protein within a    plant cell in order to prevent expression of, or knock out    expression of, said endogenous Msi2 protein within a plant cell;-   b) selecting a plant cell comprising the mutated nucleic acid    molecule, or a plant cell in which expression of said endogenous    Msi2 protein is prevented or knocked out; and-   c) optionally, regenerating a plant from said plant cell.

The invention further provides a method for making a plant as taughtherein comprising the steps of:

-   a) transforming a plant cell with a nucleic acid molecule as taught    herein, a chimeric gene as taught herein, or a vector as taught    herein, or transforming a plant cell with a nucleic acid molecule in    order to prevent expression of, or knock out expression of, an    endogenous Msi2 protein;-   b) optionally, additionally modifying in said plant cell a nucleic    acid molecule encoding an endogenous Msi2 protein in order to    prevent expression of, or knock out expression of, an endogenous    Msi2 protein;-   c) selecting a plant cell comprising the nucleic acid molecule as    taught herein, a chimeric gene as taught herein, or a vector as    taught herein, or a plant cell in which expression of endogenous    Msi2 protein is prevented or knocked out; and-   d) optionally, regenerating a plant from said plant cell.

The present invention also provides a method for making a plant astaught herein, comprising the steps of: i) transforming a plant cellwith a polynucleotide as taught herein, a chimeric gene as taughtherein, or a vector as taught herein; ii) selecting a plant cellcomprising said polynucleotide; and iii) optionally, regenerating aplant from said plant cell.

The methods for making a plant as taught herein may further comprise thestep of modifying an endogenous plant Msi2 protein-encodingpolynucleotide within said plant cell to prevent expression ofendogenous Msi2 protein.

The Msi2 protein-encoding polynucleotides, preferably a Msi2protein-encoding chimeric gene, as taught herein can be stably insertedin a conventional manner into the nuclear genome of a single plant cell,and the so-transformed plant cell can be used in a conventional mannerto produce a transformed plant that has an altered phenotype due to thepresence of the Msi2 protein as taught herein in certain cells at acertain time. In this regard, a T-DNA vector, comprising a Msi2protein-encoding polynucleotide as taught herein, in Agrobacteriumtumefaciens can be used to transform the plant cell, and thereafter, atransformed plant can be regenerated from the transformed plant cellusing the procedures described, for example, in EP 0 116 718, EP 0 270822, PCT publication WO84/02913 and published European Patentapplication EP 0 242 246 and in Gould et al. (1991, Plant Physiol.95,426-434). The construction of a T-DNA vector for Agrobacteriummediated plant transformation is well known in the art. The T-DNA vectormay be either a binary vector as described in EP 0 120 561 and EP 0 120515 or a co-integrate vector which can integrate into the AgrobacteriumTi-plasmid by homologous recombination, as described in EP 0 116 718.

Likewise, selection and regeneration of transformed plants fromtransformed plant cells is well known in the art. Obviously, fordifferent species and even for different varieties or cultivars of asingle species, protocols are specifically adapted for regeneratingtransformants at high frequency.

The resulting transformed plant can be used in a conventional plantbreeding scheme to produce haploid plants that may subsequently becomedoubled haploid plants.

Methods for the Generation of Haploid Plants and/or Doubled HaploidPlants

The invention also relates to a method of generating a haploid plant, aplant with aberrant ploidy, or a doubled haploid plant, said methodcomprising the steps of:

-   a) crossing a plant expressing an endogenous Msi2 protein to the    modified plant as taught herein, wherein the modified plant as    taught herein lacks expression of endogenous Msi2 protein at least    during embryonic development;-   b) harvesting seed;-   c) growing at least one seedling, plantlet or plant from said seed;    and-   d) selecting a haploid seedling, plantlet or plant, a seedling,    plantlet or plant with aberrant ploidy, or a doubled haploid    seedling, plantlet or plant.

Said plant expressing an endogenous Msi2 protein may be an F1 plant.

The plant expressing the endogenous Msi2 protein may be a pollen parentof the cross, or may be an ovule parent of the cross.

Crossing a modified plant as taught herein, lacking expression ofendogenous Msi2 protein, to a wild-type plant will result in at leastsome progeny that is haploid and comprises only chromosomes from theplant that expresses the endogenous Msi2 protein. Thus, the presentinvention allows for the generation of haploid plants having all of itschromosomes from a plant of interest by crossing the plant of interestwith a plant lacking expression of functional Msi2 protein, andcollecting the resulting haploid seed.

Thus, genome elimination can be engineered with a precise molecularchange independent of parental genotype. Msi2 protein is found in anyplant species. This allows haploid plants to be made in species whereconventional methods for haploid plant production, such as tissueculture of haploid cells and wide crosses, are unsuccessful.

The plant lacking expression of functional Msi2 protein as taught hereinmay be crossed as either the male or female parent. The methods taughtherein allow for transfer of paternal chromosomes into maternalcytoplasm. Thus, it can generate cytoplasmic male sterile lines with adesired genotype in a single step.

The invention further relates to a method of generating a doubledhaploid plant, said method comprising the steps of:

-   a) crossing a plant expressing an endogenous Msi2 protein to the    modified plant as taught herein, wherein the modified plant as    taught herein lacks expression of endogenous Msi2 protein at least    during embryonic development;-   b) selecting a haploid plant; and-   c) converting said haploid plant into a doubled haploid plant.

Thus, once generated, haploid plants can be used for the generation ofdoubled haploid plants, which comprise an exact duplicate copy ofchromosomes. A wide variety of methods are known for generating doubledhaploid organisms from haploid organisms. For example, chemicals such ascolchicine may be applied to convert the haploid plant into a doubledhaploid plant. Alternatively, ploidy may double spontaneously duringembryonal development or at a later developmental stage of a plant.

Doubled haploid plants can be further crossed to other plants togenerate F1, F2, or subsequent generations of plants with desiredtraits.

Doubled haploids plants may be obtained that do not bear transgenic ormutagenized genes. Additionally, doubled haploid plants can rapidlycreate homozygous F2s from a hybrid F1.

The invention also relates to a method of generating a haploid ordoubled haploid plant, said method comprising the step of identifying aplant expressing an endogenous Msi2 protein and a plant as taughtherein, wherein the plant as taught herein lacks expression ofendogenous Msi2 protein at least in its reproductive parts and/or duringembryonic development.

In an embodiment, crossing does not comprise sexually crossing the wholegenomes of plants. Instead, one set chromosomes is eliminated.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 shows a tetrad of a Msi2-K126-stop mutant. The arrow shows amicronucleus.

SEQUENCE LISTING

SEQ ID NO:1: plant Msi2 consensus protein sequence

SEQ ID NO:2: Solanum consensus Msi2 protein sequence

SEQ ID NO:3: Solanum lycopersicum Msi2 protein sequence

SEQ ID NO:4: Solanum lycopersicum Msi2_K126* coding sequence

SEQ ID NO:5: Solanum lycopersicum Msi2 coding sequence

SEQ ID NO:6: Solanum lycopersicum Msi2_K126* truncated protein sequence

SEQ ID NO:7: Solanum consensus Msi2 coding sequence

SEQ ID NO:8—Solanum lycopersicum Msi2 genomic DNA sequence

SEQ ID NO:9—Solanum lycopersicum Msi2_K126* genomic DNA sequence

SEQ ID NO:10—Solanaceae consensus Msi2 protein sequence

EXAMPLES Example 1: Uniparental Genome Elimination in Tomato Materialand Methods Plant Material

Three tomato cultivars were used namely “MoneyBerg TMV+”, “MicroTom” and“RZ52201”. From a tomato RZ52201 mutant population, following methodsdescribed in WO 2007/037678 and WO2009/041810, two somaticnon-synonymous mutants in the gene Msi2 were selected, namelyMsi2_K126-STOP and Msi2_K126M, which are both mutated at amino acidposition 126. The selected mutant plant was self-pollinated and in theoffspring, plants were selected that were homozygous for the mutatedlocus. From a tomato MoneyBerg TMV+ mutant population a somaticsynonymous mutant was selected, following methods described in WO2007/037678 and WO2009/041810, in the gene Msi2, namely Msi2_D337D,which is mutated at amino acid position 337 (C to T). The selectedmutant plant was self-pollinated and in the offspring, plants wereselected that were homozygous for the mutated locus.

Method

Uni-parental genome elimination and the resulting production of ahaploid plant was provoked by making a cross between a so called haploidinducer line and another non-haploid inducer line, for example abreeding line. Crosses of tomato lines for uni-parental genomeelimination were performed at relatively high temperatures (26-28° C.),since it is known that an elevated temperature can, but only in somecases, have a positive effect on the occurrence of uni-parental genomeelimination (Sanei et al. PNAS 108.33 (2011): E498-E505).

Results

The non-synonymous mutation of A to Tin the Msi2_K126-STOP mutantresulted in the introduction of a premature stop codon and thereby theproduction of a truncated protein (SEQ ID NO:6). The non-synonymousmutation of A to T in the Msi2_K126M mutant resulted in an amino acidmodification of a lysine to a methionine. Furthermore a SIFT analysiswas run for the Msi2 protein and the mutation of a lysine to amethionine at position 126 was rated by this analysis to be neutral(Kumar et al. Nat Protoc. 2009; 4(7):1073-81). The synonymous mutationof C to T in the Msi2_D337D mutant did not result in an amino acidmodification. Each of the three mutant plants homozygous for theMsi2_K126-STOP, the Msi2_K126M or the Msi2_D337D mutation were used aspollen donor and as female in crosses at relatively high temperatures(26-28° C.) using non-mutated wild type MicroTom plants as female orpollen donor, respectively. Table 1 lists an overview of all crossesmade and the sown seeds which were evaluated for the MicroTom phenotype.

TABLE 1 List of crosses made; genetic background of the parents used,number of offspring plants tested and number of offspring plants whichshowed MicroTom dwarf phenotype. Number of Number plants with Plant usedPlant used Background of plants MicroTom as female as male mutant parenttested phenotype Year Msi2_K126- MicroTom RZ52201 98 3 2014 STOPMicroTom Msi2_K126- RZ52201 89 2 2014 STOP Msi2_K126- MicroTom RZ52201564 4 2015 STOP MicroTom Msi2_K126- RZ52201 325 1 2015 STOP Msi2_K126MMicroTom RZ52201 19 0 2014 MicroTom Msi2_K126M RZ52201 205 0 2014Msi2_D337D MicroTom MoneyBergTMV+ 160 0 2014 MicroTom Msi2_D337DMoneyBergTMV+ 36 0 2014 RZ52201 MicroTom — 188 0 2015 MicroTom RZ52201 —188 0 2015

Seeds derived from the crosses listed in table 1 were sown and theplants were evaluated for their DNA content by means of flow cytometry.The flow cytometry analysis resulted in a determination of only normaldiploid ploidy levels for all plants tested, similar to wild type tomatocultivars such as MoneyBergTMV+. The cultivar MicroTom has a dwarfphenotype, which is known to be recessive (Marti et al, J Exp Bot, Vol.57, No. 9, pp. 2037-2047, 2006). After a cross of MicroTom to or with,for instance a MoneyBerg TMV+ or RZ52201 wild type cultivar, one onlyfinds offspring with the indeterminate non-dwarf phenotype of theMoneyBerg TMV+ or RZ52201 wild type cultivar, respectively. The same wasfound for crosses with the Msi2_D337D synonymous mutant and MicroTom;all offspring of a MicroTom and Msi2_D337D mutant crosses showed theindeterminate non-dwarf phenotype of the MoneyBerg TMV+ parent.Reciprocal crosses of MicroTom and the Msi2_K126M mutant did not resultin offspring with the MicroTom phenotype. Using the Msi2_K126-STOPmutant as male or female parent, in total 10 plants were found whichshowed a MicroTom phenotype. This indicates that the RZ52201 parentgenetic material is not part of the resulting offspring and thisindicates that these 10 offspring plants are of haploid MicroTom origin.The ploidy of all plants of the latter 10 plants was found to bediploid, indicating that spontaneous doubling had occurred, a phenomenawhich has been described to have an exceptional high frequency ofappearance for tomato (Report of the Tomato Genetics Cooperative Number62-December 2012).

In order to determine whether and to what extent uni-parental genomeelimination had occurred, a single nucleotide polymorphism (SNP) assaywas run for in total 44 positions for the 2014 offspring, spread acrosseach of the 12 tomato chromosomes (4 SNPs on chromosome 1, 2, 3, 4, 5,6, 11 and 12; 3 SNPs on chromosome 8 and 10; 2 SNPs on chromosome 9).The same analysis was performed for the 2015 offspring, now on 22positions (2 SNPs on chromosome 1, 2, 3, 4, 5, 6, 7, 8, 10 and 12; 1 SNPon chromosome 9 and 11). The single 5 nucleotide polymorphisms selectedwere homozygous for one base pair for the MicroTom parent and homozygousfor all but not the MicroTom base pair in the RZ52201 parent. A regularcross between a wild type MicroTom cultivar and the RZ52201 cultivarwould result in a heterozygous single nucleotide polymorphism score.However, when the process of uniparental genome elimination hasoccurred, one expects the loss of the haploid inducer line genome. Thesingle nucleotide polymorphism test resulted in calling of onlyhomozygous base pair scores from the MicroTom parent for each of the 5offspring plants which also showed the MicroTom phenotype and none ofthe RZ52201 parent were called. Based on the single nucleotidepolymorphism scores it was concluded that the complete genome of theMsi2_K126-STOP mutant was no longer present in the offspring. Therefore,it can be concluded that the Msi2_K126-STOP mutant functions as a highlyefficient haploid inducer line. In the crosses in which theMsi2_K126-STOP mutant was used as female parent, a selfing of MicroTomcan be ruled out. It is highly unlikely that in the experiment usingMicroTom as female parent selfing took place, given the very low numberof offspring showing the MicroTom phenotype (only 2 seeds out of 89 and1 seed out of 325), and the fact that only homozygous base pairs werescored.

Pollen tetrads of the Msi2-K126-stop mutant and of RZ52201 controlplants were checked for occurrence of aberrancies. From four differentflower trusses at least two flowers were taken and anthers were squashedin order to look at pollen tetrads. For the Msi2-K126-stop mutant, inall 10 observed anthers from 5 individual flowers, micronuclei wereobserved (see FIG. 1 ). In each observed anther several examples ofmicronuclei were found, however not more than 1% of the pollen tetradsin an anther showed the aberrancies. For the RZ52201 control, rarely ananther was observed containing pollen tetrads with micronuclei. Twoanthers were found in which in total only two or three examples ofmicronuclei could be observed. In a second round of experiments, themicronuclei were counted; For the Msi2-K126-stop mutant, micronucleiwere observed with a frequency of 1.94% (n=1). For the RZ52201 controlplant flowers, micronuclei were observed with an average frequency of0.58±0.36% (n=5). It is concluded that the separation of chromosomesduring meiosis is considerably more frequently disturbed as a result onthe Msi2-K126-stop mutation compared to the control. Aberrant mitosis,for instance observations of micronuclei, are often used as directevidences of chromosome elimination and haploid production in inter-,intra-specific hybridizations in crops. For example, aberrant mitosis aswell as aberrant meiosis, for instance micronuclei, were found in astudy of a maize DH-inducer line (Qiu, Fazhan, et al. Current PlantBiology 1 (2014): 83-90). The observations of meiosis micronuclei in theMsi2-K126-stop mutant, suggest that during mitosis similar processesoccur. It is likely that the process of uniparental genome eliminationduring the first mitotic divisions after fusion of wild type andMsi2-K126-stop zygotes takes place and that this results in the observedinduction of haploids.

Example 2: Uniparental Genome Elimination in Arabidopsis Materials andMethods Plant Material

The following Arabidopsis NASC stock centre accessions were used;Columbia (background line, Col-0, N1092), Col-5 (N1644), ArabidopsisMsi2 gene (At2g16780) T-DNA insertion lines (N720344 and N501214, inCol-0 background) and, since it is not known whether the Msi2 and Msi3gene are functionally redundant genes, Arabidopsis Msi3 gene (At4g35050)T-DNA insertion mutants (N309860, N309863 and N564092 in Col-0background). The T-DNA insertion lines were evaluated by means of PCRamplification and subsequent sequencing of the putative T-DNA insertionlocus in order to determine the exact insertion in the Arabidopsis Msi2and Msi3 genes. Based on the finding that the insertions were located inexons of either Msi2 or Msi3 genes it was concluded that these T-DNAinsertion lines are true knock-outs for either the Msi2 or Msi3 gene.The exact positions as counted in number of bases downstream of thestart codon were; N720344 (position 429, exon 2), N501214 (position 111,exon 1), N309863/N309860 (both in position 559, exon 2) and N564092(position 1237, exon 6). By making crosses between two insertion linesand selecting for homozygous T-DNA insertions in both the Msi2 and theMsi3 gene, two novel double T-DNA insertion lines were produced;N720344+N309860 and N309863+N501214.

Method

Uni-parental genome elimination and the resulting production of ahaploid plant is provoked by making a cross between a so called haploidinducer line and another non-haploid inducer line, for example aColumbia background (Col-0) control line.

Results

Either a single T-DNA insertion line for Msi2 (N720344 and N501214), aMsi3 T-DNA insertion line (N309863 and N564092), the two newly generatedMsi2/Msi3 double T-DNA insertion lines (N720344+N309860 andN309863+N501214) or Col-0 background plants are used as pollen donor andas female in crosses using Col-5 as female or pollen donor,respectively. Table 2 lists an example of typical crosses which can bemade and an example of the evaluation of the offspring for the Col-5phenotype. Col-5 has a clear distinct recessive phenotype compared tothe T-DNA insertion lines and Col-0, namely trichomeless leaves.

TABLE 2 List of crosses which can be made; genetic background of allinsertion lines was Col-0, and number of offspring plants which aretested. Plant used as Plant used as Number of female male plants testedMsi2 (N720344) Col-5 300 Col-5 Msi2 (N720344) 300 Msi2 (N501214) Col-5300 Col-5 Msi2 (N501214) 300 Msi3 (N309863) Col-5 300 Col-5 Msi3(N309863) 300 Msi3 (N564092) Col-5 300 Col-5 Msi3 (N564092) 300Msi2/Msi3 Col-5 300 (N720344 + N309860) Col-5 Msi2/Msi3 300 (N720344 +N309860) Msi2/Msi3 Col-5 300 (N309863 + N501214) Col-5 Msi2/Msi3 300(N309863 + N501214) Col-0 Col-5 300 Col-5 Col-0 300

The Col-5 accession harbours the gl1-1/gl1-1 locus giving it atrichomeless phenotype, which is known to be recessive (Kuppu et al.PLoS Genet 11.9 2015 e1005494). After a cross of Col-5 to or with, forinstance a Col-0 wild type cultivar, one only finds offspring withtrichomes coming from the dominant Col-0 allele. Using Msi2 single, Msi3single or Msi2/Msi3 double T-DNA insertion lines as male or femaleparent, in total several plants are found which show a trichomelessphenotype. This indicates that the Col-0 parent genetic material is notpart of the resulting offspring and this indicates that these offspringplants are of haploid Col-5 origin.

Based on the single Col-5 phenotype individuals among the offspring ofthe crosses performed, it is concluded that the complete genome of therespective T-DNA insertion line in the Col-0 background is no longerpresent in the offspring. Therefore, it is concluded that the T-DNAinsertion lines “N720344, N501214, N309863, N564092, N720344+N309860(double insertion line) and N309863+N501214 (double insertion line)function as highly efficient (doubled) haploid inducer lines.

The invention claimed is:
 1. A Msi2 gene comprising a loss-of-expressionmutation, wherein the Msi2 gene is derived from a gene encoding an Msi2protein having at least 70% sequence identity with at least one of SEQID NO. 1, 2, 3 and 10, wherein the loss-of-expression mutation resultsin reduced expression of the Msi2 protein, wherein the gene is not anArabidopsis thaliana-derived Msi2 gene, and wherein the Msi2 gene, whenpresent in a plant in the absence of the endogenous Msi2 gene, allowsgeneration of haploid progeny, or progeny with aberrant ploidy, at amore than normal frequency when the plant is crossed with a wild-typeplant.
 2. The gene according to claim 1, wherein the sequence encodingthe Msi2 protein has at least 90% sequence identity with at least one ofSEQ ID NO: 5 and
 7. 3. The gene according to claim 1, wherein theexpression of the Msi2 protein is knocked out.
 4. The gene according toclaim 1, wherein the loss-of-expression mutation is in a transcriptionregulation sequence.
 5. The gene according to claim 1, wherein themutation is at least one of a frame-shift mutation, deletion,substitution, rearrangement or insertion of nucleotides in theMsi2-coding region.
 6. The gene according to claim 1, herein at least10% of the Msi2-coding region is removed.
 7. A plant, seed, or plantcell comprising the Msi2 gene of claim
 1. 8. The plant, seed or plantcell according to claim 7, wherein the expression of an endogenous Msi2protein is reduced or knocked out.
 9. The plant, seed or plant cellaccording to claim 7, which is a Solanum plant, seed or plant cell. 10.The plant, seed or plant cell according to claim 8, wherein the Solanumplant, seed or plant cell is a Solanum lycopersicum.
 11. A method forproducing a plant, seed, or plant cell, the method comprising: (a)modifying an endogenous Msi2 gene within a plant cell to obtain themutated Msi2 gene, wherein the gene comprises a loss-of-expressionmutation resulting in a reduced expression of the Msi2 protein, andwherein the gene is not an Arabidopsis thaliana-derived Msi2 gene; (b)selecting a plant cell comprising the mutated Msi2 gene; and (c)optionally, regenerating a plant from said plant cell.
 12. A method ofgenerating a haploid plant, a plant with aberrant ploidy, or a doubledhaploid plant, comprising: (a) crossing a plant expressing an endogenousMsi2 protein with a plant of claim 7 wherein the plant lacks expressionof endogenous Msi2 protein at least in its reproductive parts and/orduring embryonic development; (b) harvesting seed; (c) growing at leastone seedling, plantlet or plant from said seed; and (d) selecting ahaploid seedling, plantlet or plant, a seedling, plantlet or plant withaberrant ploidy, or a doubled haploid seedling, plantlet or plant.
 13. Amethod of generating a doubled haploid plant, comprising: (a) crossing aplant expressing an endogenous Msi2 protein to the plant of claim 7,wherein the plant lacks expression of endogenous Msi2 protein at leastin its reproductive parts and/or during embryonic development; (b)selecting a haploid plant; and (c) converting said haploid plant into adoubled haploid plant.
 14. The method according to claim 13, wherein theconversion in (c) is performed by treatment with colchicine.